Methods for culturing and/or differentiating hematopoietic stem cells into progenitors and uses thereof

ABSTRACT

The present description relates to in vitro methods for culturing hematopoietic stem cells (HSCs) under mild hyperthermia conditions (e.g., between 38° C. and 40° C.) in the presence of a pyrimidoindole derivative agonist of hematopoietic stem cell expansion. The combined use of mild hyperthermia and the pyrimidoindole derivative act synergistically to promote expansion of CD34+ HSCs and/or differentiation into progenitor cells (e.g., megakaryocytic progenitors). The present description also relates to in vitro expanded cell populations of HSCs and/or progenitors, as well as uses thereof in therapy (e.g., transplantation).

The present description relates to hematopoietic stem cells. Moreparticularly, the present description relates to culturing hematopoieticstem cells under mild hyperthermia (e.g., between 38° C. and 40° C.) andin the presence of a pyrimidoindole derivative agonist of hematopoieticstem cell expansion.

BACKGROUND

Hematopoietic stem cells (HSCs) are derived from the mesoderm and areresponsible for the production of all cellular components found inblood, whether of the myeloid or lymphoid lineages. HSCs differentiateinto common myeloid progenitors (CMPs) and common lymphoid progenitors(CLPs). CMPs give rise to cells of the erythroid, granulocytic,monocytic, megakaryocytic, and dendritic lineages, whereas CLPs lead tothe derivation of T and B lymphocytes, plasma cells, natural killercells and lymphoid dendritic cells. Terminal differentiation of myeloidlineage cells ultimately leads to the generation and renewal of redblood cells, granulocytes, monocytes, myeloid-derived dendritic cells,and platelets. Additionally, HSCs (primitive or long term) have thecapacity for self-renewal, thereby ensuring an adequate supply ofprogenitors and terminally differentiated blood cells for the entirelifetime of an individual. The ability of these cells to self-renew hasled to major advances in the medical field, namely, HSC transplantationas a treatment modality in patients suffering from hematological cancersor bone marrow failure. Historically, the bone marrow has been theprimary source of HSCs, and remains to this day an important source ofcells for bone marrow replacement. Since then, umbilical cord blood andperipheral blood from G-CSF-mobilized donors have also been used as HSCsources.

In parallel with their use in the clinic, HSCs have been the subject ofintense research efforts aimed at the in vitro culture, expansion,differentiation into cells of various lineages, and the production ofblood components in the laboratory. This research led to the discoveryof several lineage-specific cytokines which nowadays are routinely usedin various culture methods allowing the preferential growth and/ordifferentiation of hematopoietic stem cells towards specific lineages.Additionally, it was discovered that culturing HSCs under fever-likemild hyperthermia (39° C. instead of the standard temperature of 37° C.)leads to an accelerated expansion and differentiation towards themegakaryocytic lineage in the presence of a megakaryocyte-inducingcytokine cocktail (Proulx et al., 2004). More recently, a class ofpyrimidoindole compounds was identified for its capacity to expandlong-term hematopoietic stem cells (Fares et al, 2014).

Despite these advances, there are a number of shortcomings to the invitro expansion of HSCs and their preferential differentiation towardsspecific myeloid lineages. For example, it is still particularlydifficult to expand HSCs in vitro, while maintaining a primitivephenotype and controlled differentiation. Efficient expansion andmaintenance of cells into a specific lineage also remain a challenge.For example, the levels of expansion required to envision the in vitroproduction of blood components remains insufficient with today'stechnology. In addition, current culture methods generally give rise toheterogeneous cellular populations that are not amenable to medicalapplications for specific clinical indications. The in vitro productionof specific populations of stem cells and progenitors will requiresubstantial improvements in order to enable their use in the clinic.There is thus a need for methods and technologies enabling massive invitro expansion, while simultaneously allowing control of the directionand extent of differentiation of hematopoietic stem and progenitor cellsinto homogeneous cell populations for clinical use and mass production.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY

The present description arises from the surprising discovery thathematopoietic stem cells (HSCs) may be advantageously cultured undermild hyperthermia (e.g., between 38° C. and 40° C.) and in the presenceof a pyrimidoindole derivative agonist of hematopoietic stem cellexpansion. The combined use of mild hyperthermia and the pyrimidoindolederivative are shown herein to act synergistically to promote expansionof CD34+ HSCs (including long-term HSCs) and/or differentiation into“lineage-primed” or progenitor cells, and/or their maintenance (e.g.,myeloid progenitors, megakaryocytic progenitors).

Indeed, in some embodiments, the combination of mild hyperthermia and apyrimidoindole derivative agonist of hematopoietic stem cell expansionwas shown to exponentially expand HSCs (CD34+ cells) when culture mediafavoring hematopoietic stem cell self-renewal were used. Without beingbound by theory, the results disclosed herein suggest that mildhyperthermia combined with the use of a pyrimidoindole compoundpreserves the anti-differentiation effects of the pyrimidoindolecompound, while simultaneously potentiating the stimulatory effects ofthe mild hyperthermia incubation temperature.

In other embodiments, the combination of mild hyperthermia and apyrimidoindole derivative agonist of hematopoietic stem cell expansionwas also shown to exponentially increase differentiation of HSCs intoprogenitor cells (e.g., myeloid progenitors, megakaryocyticprogenitors), when culture conditions that favor differentiation towardsthe megakaryocytic lineage were used. Without being bound by theory, theresults disclosed herein suggest that the pyrimidoindole compound blocksthe hyperthermia-induced differentiation of HSCs before the maturemegakaryocyte stage, and/or preferentially expands and maintainsmegakaryocytic progenitors. Thus, in some embodiments, thepyrimidoindole compound may be removed and the cells may be furtherpropagated under conditions of mild hyperthermia, thereby producing amore synchronized cell population.

Accordingly, the present description may relate to the followingaspects:

1. An in vitro method for culturing hematopoietic stem cells, saidmethod comprising:

-   -   (a) propagating said hematopoietic stem cells in a cell culture        medium comprising a pyrimidoindole derivative agonist of        hematopoietic stem cell expansion; and    -   (b) incubating said hematopoietic stem cells at an incubation        temperature between 38° C. and 40° C.        2. The method of aspect 1, wherein said hematopoietic stem cells        are CD34+ hematopoietic stem cells.        3. The method of aspect 1 or 2, wherein said hematopoietic stem        cells are from:    -   (a) umbilical cord blood;    -   (b) bone marrow;    -   (c) peripheral blood;    -   (d) induced pluripotent stem cells;    -   (e) embryonic stem cells;    -   (f) transdifferentiated from differentiated cells of        non-hematopoietic origin;    -   (g) genetically modified hematopoietic stem cells;    -   (h) immortalized hematopoietic stem cells;    -   (i) other sources of pluripotent or multipotent cells; or    -   (j) any combination thereof.        4. The method of aspect 3, wherein said hematopoietic stem cells        are from mobilized peripheral blood cells.        5. The method of aspect 3, wherein said hematopoietic stem cells        are from unmobilized peripheral blood cells.        6. The method of aspect 4 or 5, wherein said hematopoietic stem        cells are from residual cells following leukoreduction,        deleukocytation, and/or other blood purification or processing        of peripheral blood.        7. The method of any one of aspects 1 to 6, wherein said        hematopoietic stem cells are incubated in a cell culture medium        comprising a pyrimidoindole derivative agonist of hematopoietic        stem cell expansion, and/or at an incubation temperature between        38° C. and 40° C., for at least 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, or 21 days.        8. The method of any one of aspects 1 to 7, wherein said        incubation temperature is 39° C.        9. The method of any one of aspects 1 to 8, wherein said        pyrimidoindole derivative agonist of hematopoietic stem cell        expansion is:    -   (a) a pyrimido[4,5-b]indole derivative;    -   (b)        (1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine;    -   (c) methyl        4-(3-(piperidin-1-yl)propylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate;    -   (d) methyl        4-(3-(piperidin-1-yl)propylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate        hydrochloride;    -   (e) a pharmaceutically acceptable salt, solvate, prodrug, or        stereoisomer of any one of (a) to (d); or    -   (f) any combination of (a) to (e).        10. The method of any one of aspects 1 to 9, wherein said cell        culture medium comprises stem cell factor (SCF); thrombopoietin        (TPO); or both SCF and TPO.        11. The method of any one of aspects 1 to 9, wherein said method        is for expanding hematopoietic stem cells, and wherein said cell        culture medium is a hematopoietic stem cell culture medium.        12. The method of aspect 11, wherein said hematopoietic stem        cell culture medium comprises: human FMS-like tyrosine kinase 3        ligand (FLT3); stem cell factor (SCF); thrombopoietin (TPO);        low-density lipoprotein (LDL); or any combination thereof.        13. The method of any one of aspects 1 to 9, wherein said method        is for culturing hematopoietic stem cells to produce        megakaryocytic progenitor cells, and wherein said cell culture        medium is a medium promoting differentiation of hematopoietic        stem cells towards the megakaryocytic lineage.        14. The method of aspect 13, wherein said medium promoting        differentiation of hematopoietic stem cells towards the        megakaryocytic lineage comprises: stem cell factor (SCF);        thrombopoietin (TPO); human FMS-like tyrosine kinase 3 ligand        (FLT3); IL-6; IL-9; or any combination thereof.        15. The method of any one of aspects 1 to 9, wherein said method        is for culturing hematopoietic stem cells to produce myeloid        progenitor cells, and wherein said cell culture medium is a        medium promoting differentiation of hematopoietic stem cells        towards the myeloid progenitor cell lineage.        16. The method of any one of aspects 1 to 15, further        comprising: (c) removing said pyrimidoindole derivative agonist        of hematopoietic stem cell expansion and continuing to propagate        said cells at an incubation temperature between 38° C. and        40° C. or at an incubation temperature of about 37° C., thereby        synchronizing said cells.        17. An in vitro expanded cell population which is:    -   (a) a population of expanded hematopoietic stem cells produced        by the method of aspect 11 or 12;    -   (b) a population of megakaryocytic progenitor cells:        -   (i) produced by the method of aspect 13 or 14; and/or        -   (ii) comprising at least 40%, 45%, 50%, 55%, 60%, 65%, 70%,            75%, or 80% of CD34+/CD41+ cells;    -   (c) a population of myeloid progenitor cells produced by the        method of aspect 15;    -   (d) a synchronized cell population produced by the method of        aspect 16; or    -   (e) any combination of (a) to (d).        18. The in vitro expanded cell population of aspect 17 for use        in transplantation in a subject.        19. The in vitro expanded cell population of aspect 17, wherein:    -   (a) said population of expanded hematopoietic stem cells is for        use in hematopoietic stem cell transplantation, or for the        manufacture of a therapeutic composition for same;    -   (b) said population of megakaryocytic progenitor cells is for        use in the treatment of thrombocytopenia, or for the manufacture        of a therapeutic composition for same; or    -   (c) said population of myeloid progenitor cells is for use in        myeloid progenitor cell transplantation, or for the manufacture        of a therapeutic composition for same.

20. Use of:

-   -   (a) the population of expanded hematopoietic stem cells of        aspect 17 for hematopoietic stem cell transplantation;    -   (b) the population of megakaryocytic progenitor cells of aspect        17 for the treatment of thrombocytopenia; or    -   (c) the population of myeloid progenitor cells of aspect 17 for        myeloid progenitor cell transplantation.        21. A pharmaceutical composition comprising:    -   (a) the population of expanded hematopoietic stem cells of        aspect 17;    -   (b) the population of megakaryocytic progenitor cells of aspect        17; or    -   (c) the population of myeloid progenitor cells of aspect 17.

General Definitions

Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., arepresented merely for ease of reading the specification and claims. Theuse of headings or other identifiers in the specification or claims doesnot necessarily require the steps or elements be performed inalphabetical or numerical order or the order in which they arepresented.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one” butit is also consistent with the meaning of “one or more”, “at least one”,and “one or more than one”.

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. In general, the terminology “about” is meant to designate apossible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4,5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unlessindicated otherwise, use of the term “about” before a range applies toboth ends of the range.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, un-recitedelements or method steps.

Other objects, advantages and features of the present description willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 shows the fold expansion of CD34+/CD41+ megakaryocytic progenitorcells as a function of time for CD34+ cells cultured under conditionsthat promote differentiation towards the megakaryocytic lineage. Fourconditions were tested: (1) “37° C.”: at standard temperature in theabsence of a pyrimidoindole derivative agonist of hematopoietic stemcell expansion (PIC); (2) “37° C.+PIC”: at standard temperature in thepresence of 35 nM PIC; (3) “39° C.”: under mild hyperthermia in theabsence of PIC; or (4) “39° C.+PIC”: under mild hyperthermia in thepresence of 35 nM PIC.

FIG. 2 shows the number of megakaryocytic colony-forming units (CFU-MK)produced under the four culture conditions tested in FIG. 1, asdetermined by a standard CFU-MK assay.

FIG. 3(A) shows fold expansion of CD34+ hematopoietic stem cells as afunction of time for CD34+ cells cultured under conditions that promotehematopoietic stem cell expansion while limiting differentiation. Fourconditions were tested: (1) “37° C.”: at standard temperature in theabsence of a pyrimidoindole derivative agonist of hematopoietic stemcell expansion (PIC); (2) “37° C.+PIC”: at standard temperature in thepresence of 35 nM PIC; (3) “39° C.”: under mild hyperthermia in theabsence of PIC; or (4) “39° C.+PIC”: under mild hyperthermia in thepresence of 35 nM PIC. FIG. 3(B) shows fold expansion of CD34+/CD45RA−long-term hematopoietic stem cells (LT-HSCs) as a function of timestarting with an initial population of purified CD34+ cells culturedunder conditions that promote hematopoietic stem cell expansion whilelimiting differentiation. Four conditions were tested: (1) “37° C.”: atstandard temperature in the absence of a pyrimidoindole derivativeagonist of hematopoietic stem cell expansion (PIC); (2) “37° C.+PIC”: atstandard temperature in the presence of 35 nM PIC; (3) “39° C.”: undermild hyperthermia in the absence of PIC; or (4) “39° C.+PIC”: under mildhyperthermia in the presence of 35 nM PIC.

FIGS. 4-6 show the fold expansion of CD34+/CD41+ cells (megakaryocyticprogenitors) over 14 days in the presence (“39”) or absence (“37”) ofmild hyperthermia and/or the presence or absence of PIC. Expansions wereperformed from cord blood CD34+ cells (FIG. 4), mobilized peripheralblood CD34+ cells (FIG. 5), or bone marrow CD34+ cells (FIG. 6).

FIG. 7 shows the fold expansion of CD34+/CD41+ cells (megakaryocyticprogenitors) over 14 days in the presence (“39”) or absence (“37”) ofmild hyperthermia and/or the presence or absence of anotherpyrimidoindole derivative agonist of hematopoietic stem cell expansion(PIC2).

FIGS. 8 and 9 show the results of human platelet production at 5 daysand 2.5 weeks, respectively, in immunodeficient mice transplanted withhuman cord blood CD34+ cells expanded in vitro under conditions of mildhyperthermia and in a culture medium favoring the preferential expansionof megakaryocyte (MK) progenitors (CD41+ cells). “MK-6M”: infusion ofsix million CD41+ cells produced in the absence of PIC; “(MK+PIC)-1M”:infusion of one million CD41+ cells produced in the presence of PIC;“(MK+PIC)-6M”: infusion of six million CD41+ cells produced in thepresence of PIC; and “PBS”: infusion of phosphate-buffered saline as acontrol. The numbers to the right of the dash symbols represent meannumbers of human platelets/μL of blood for each condition.

FIG. 10 shows the fold expansion of cord blood-derived CD71+ cells(erythroid precursors) over 14 days in the presence (“39”) or absence(“37”) of mild hyperthermia and/or the presence or absence of PIC.

FIG. 11 shows the fold expansion of total cells (total cell expansion)over 14 days in the presence (“39”) or absence (“37”) of mildhyperthermia and/or the presence or absence of PIC.

FIGS. 12 and 13 show the fold expansion of CD34+ cells (HSGs) andCD34+/CD71+ cells (erythroid progenitors), respectively, over 14 days inthe presence (“39”) or absence (“37”) of mild hyperthermia and/or thepresence or absence of PIC.

FIG. 14 shows the fold-expansion of cord blood-derived CD34+ cells(hematopoietic stem cells) cultured in medium supplemented with eitherthe commercially available CC110 cytokine cocktail (FIG. 14A) or ahome-made (HM) cytokine cocktail (FIG. 14B). The cells were cultured inthe presence (“39”) or absence (“37”) of mild hyperthermia and/or thepresence or absence of PIC.

FIG. 15 shows the fold-expansion of cord blood-derived CD34+/CD45RA−(long-term hematopoietic stem cells; LT-HSGs) cells cultured in mediumsupplemented with either the CC110 cytokine cocktail (FIG. 15A) or thehome-made (HM) cytokine cocktail (FIG. 15B). The cells were cultured inthe presence (“39”) or absence (“37”) of mild hyperthermia and/or thepresence or absence of PIC.

FIG. 16 shows the fold-expansion of cord blood-derived CD34+ cells(hematopoietic stem cells) cultured in StemSpan™ SFEM mediumsupplemented with either the CC110 cytokine cocktail (FIG. 16A) or thehome-made (HM) cytokine cocktail (FIG. 16B). The cells were cultured inthe presence (“39”) or absence (“37”) of mild hyperthermia and/or thepresence or absence of PIC.

FIG. 17 shows the fold-expansion of cord blood-derived CD34+ cells(hematopoietic stem cells) cultured in StemSpan™ SFEM II mediumsupplemented with either the CC110 cytokine cocktail (FIG. 17A) or thehome-made (HM) cytokine cocktail (FIG. 17B). The cells were cultured inthe presence (“39”) or absence (“37”) of mild hyperthermia and/or thepresence or absence of PIC.

FIG. 18 shows the fold-expansion of cord blood-derived CD34+ cells(hematopoietic stem cells) cultured in StemSpan™ ACF medium supplementedwith either the CC110 cytokine cocktail (FIG. 18A) or the home-made (HM)cytokine cocktail (FIG. 18B). The cells were cultured in the presence(“39”) or absence (“37”) of mild hyperthermia and/or the presence orabsence of PIC. FIG. 18C shows the fold-expansion of CB CD34+/CD45RA−cells (long-term hematopoietic stem cells; LT-HSC) cultured in StemSpan™SFEM medium supplemented with the BS1 cytokine cocktail under mildhyperthermia and in the presence (“39”) or absence (“CTL”) of PIC.

FIGS. 19, 20 and 21 show the fold-expansion of CD34+ cells(hematopoietic stem cells), CD34+/CD45RA− cells (LT-HSGs), andCD34+/CD38−/CD45RA− cells, respectively, after 7, 10, and 14 days inculture in the presence (“39”) or absence (“37”) of mild hyperthermiaand/or the presence or absence of PIC2.

FIG. 22 shows the fold-expansion of CD34+/CD38−/CD45RA− cells after 14days of culture under mild hyperthermia (39° C.) and PIC.

FIGS. 23, 24 and 25 show the fold-expansion of mobilized peripheralblood-derived CD34+ cells (hematopoietic stem cells), CD34+/CD45RA−cells (LT-HSGs), and CD34+/CD38−/CD45RA− cells, respectively, after 10,14, and 17 days in culture in the presence (“39”) or absence (“37”) ofmild hyperthermia and/or the presence or absence of PIC.

FIGS. 26, 27 and 28 show the fold-expansion of human bone marrow-derivedCD34+ cells (hematopoietic stem cells), CD34+/CD45RA− cells (LT-HSGs),and CD34+/CD38−/CD45RA− cells, respectively, after 10, 14, and 17 daysin culture in the presence (“39”) or absence (“37”) of mild hyperthermiaand/or the presence or absence of PIC.

FIG. 29 shows the fold-expansion of CD34+/ALDH^(Bright) cells over 21days in culture in the presence (“39”) or absence (“37”) of mildhyperthermia and/or the presence or absence of PIC.

DETAILED DESCRIPTION

The present description arises from the surprising discovery that cellculture conditions comprising mild hyperthermia (e.g., between 38° C.and 40° C.) and the presence of a pyrimidoindole derivative agonist ofhematopoietic stem cell expansion, act synergistically to promoteexpansion of hematopoietic stem cells (HSGs) and/or differentiation intoprogenitor cells (e.g., myeloid progenitors, megakaryocyticprogenitors). Indeed, in some embodiments, the combination of mildhyperthermia and of a pyrimidoindole derivative agonist of hematopoieticstem cell expansion was shown to exponentially expand hematopoietic stemcells. In addition, this combination was also shown, in someembodiments, to exponentially increase differentiation of HSGs intoprogenitor cells (e.g., myeloid progenitors, megakaryocyticprogenitors).

In some aspects, the present description relates to an in vitro methodfor culturing hematopoietic stem cells, the method comprising: (a)propagating said hematopoietic stem cells in a cell culture mediumcomprising a pyrimidoindole derivative agonist of hematopoietic stemcell expansion; and (b) incubating said hematopoietic stem cells underconditions of mild hyperthermia (e.g., at an incubation temperaturebetween 38° C. and 40° C.). In some embodiments, the method may furthercomprise: (c) removing the pyrimidoindole derivative agonist ofhematopoietic stem cell expansion and continuing to propagate the cellsat an incubation temperature between 38° C. and 40° C., therebyproducing a more synchronized cell population.

As used herein, “hematopoietic stem cells” or “HSCs” refers to cellspossessing both multipotency (i.e., can produce all cellular componentsfound in blood, such as leukocytes, erythrocytes, platelets, etc.), andthe ability to self-renew. The use of CD34 as a positive selectionmarker allows to enrich for hematopoietic stem cells, and thus, in someembodiments, CD34+ cells may be used as hematopoietic stem cells. Inaddition to CD34, a plurality of other hematopoietic stem cell markerscan be used alone or in combination, to identify, enrich and/or isolatea given population of HSCs by methods known in the art (e.g., FACS,immunomagnetic particles). Examples of other markers that may be usefulfor this purpose include CD133/AC133+, Lin−, ALDH^(hi) or ALDH^(Bright),CD38−, CD71−, HLA-DR−, CD33−, CD117/c-kit+, CD59+, CD90/Thy-1+, andCD49f+. In particular, in some embodiments, long-term hematopoietic stemcells (LT-HSCs) may be identified, enriched and/or isolated usingmarkers such as CD34+/CD45RA−, CD34+/CD38−/CD45RA−, and CD49f+. Othermarkers not listed here may also be useful for identifying, enrichingand/or isolating a given population of HSCs, and such markers are alsoconsidered within the scope of the present description. In someembodiments, the HSCs may be isolated or derived from cells of umbilicalcord blood; bone marrow; peripheral blood; induced pluripotent stemcells; embryonic stem cells; cells obtained by transdifferentiation ofnon-hematopoietic, differentiated cells; HSCs that have been geneticallymodified; HSCs that have been immortalized or engineered; other sourcesof pluripotent or multipotent cells; or any combination thereof. In someembodiments, the HSCs may be isolated or derived from mobilizedperipheral blood cells (e.g. mobilized with G-CSF). In some embodiments,the HSCs may be isolated or derived from unmobilized peripheral bloodcells (i.e., peripheral blood cells that have not been mobilized with,for example, G-CSF). In some embodiments, the HSCs may be isolated orderived from cells remaining after the leukoreduction and/ordeleukocyration of mobilized or unmobilized peripheral blood cells(e.g., cells remaining in the leukoreduction chamber of aplateletpheresis apparatus, or cells recovered from leukoreductionfilters of whole blood collection sets; these leukoreduction devices areotherwise discarded). It should be understood that the presentdescription may be applied to HSCs from diverse sources and methods ofpurification, treatment, and/or concentration. Non limiting examples ofsuch sources and methods are described herein, but other sources of HSCsnot explicitly mentioned herein may also be used in accordance with thepresent description.

As used herein, the term “propagating” refers to the in vitro culture ofcells (e.g., hematopoietic stem cells) to achieve a particular purpose,such as expanding a certain cell population (e.g., HSCs and/or CD34+cells), and/or promoting cell differentiation towards a desired celllineage (e.g., megakaryocytic lineage, or other lineages arising frommyeloid progenitor cells). In some embodiments, “propagating” may referto small “lab-scale” cell cultures or larger-scale cell cultures (e.g.,using bioreactors). In some embodiments, the composition of the cellculture medium used for propagating the cells may be selected to promoteexpansion of a particular cell type (e.g., HSCs and/or CD34+ cells), orto promote differentiation towards a desired cell lineage (e.g.,megakaryocytic lineage, or other lineages arising from myeloidprogenitor cells). In some embodiments, the cell culture medium of thepresent description may comprise one or more cytokines. In someembodiments, the cytokines may comprise: stem cell factor (SCF);thrombopoietin (TPO); or both. As used herein, the expression “promotedifferentiation towards” refers to the general direction of cellulardifferentiation towards a particular endpoint, but not necessarily tothe reaching of the endpoint (i.e., final differentiation of a cell intoa mature, fully differentiated cell). Such media and additives (e.g.,different cytokine cocktails and/or other cell culture media componentsto influence expansion and/or differentiation towards particularlineages) are generally known to the person skilled in the art. Many ofthe suitable media and additives are commercially available to theskilled person.

For example, for CD34+ cell culture and expansion with limiteddifferentiation in the context of the present description, cells may bepropagated in a variety of commercially available media such asStemSpan™ ACF, SFEM, or SFEM II, and supplemented with a commerciallyavailable cytokine cocktail such as CC110 (STEMCELL Technologies,Vancouver, BC, Canada). In some embodiments, a home-made (HM) cytokinecocktail may be prepared which may comprise: human FMS-like tyrosinekinase 3 ligand (FLT3) (e.g., 100 ng/mL), stem cell factor (SCF) (e.g.,100 ng/mL), thrombopoietin (TPO) (e.g., 50 ng/mL), low-densitylipoprotein (LDL) (e.g., 10 μg/mL), or any combination thereof. Suchcell culture media/cytokine cocktails and others, which enable thepropagation of HSCs (e.g., CD34+ cells) under self-renewal conditions inthe context of the present description, are herein referred to as“hematopoietic stem cell culture medium”. However, other types of cellculture media and/or additives (e.g., cytokine cocktails) may be usedand the present description should not be limited to the cell culturemedia employed in the present Examples.

For culture and differentiation towards the megakaryocytic lineage inthe context of the present description, purified CD34+ cells may becultured in a medium such as StemSpan™ ACF or SFEM medium (STEMCELL™Technologies), supplemented with a cytokine cocktail such as the OMPCcytokine cocktail described in Robert et al., 2011, or the BS1megakaryocyte expansion and differentiation cytokine cocktail describedin Cortin et al., 2005. The OMPC cytokine cocktail includes TPO (e.g.,35 ng/mL), SCF (e.g., 10 ng/mL), and human FLT3 (11 ng/mL). The BS1cytokine cocktail includes TPO (e.g., 30 ng/mL TPO), SCF (1 ng/mL), IL-6(e.g., 7.5 ng/mL), and IL-9 (e.g., 13.5 ng/mL). Such cell culturemedia/cytokine cocktails and others, which promote the differentiationof HSCs towards the megakaryocytic lineage in the context of the presentdescription, are herein referred to as “medium promoting differentiationof hematopoietic stem cells towards the megakaryocytic lineage”.However, other types of cell culture media and/or additives (e.g.,cytokine cocktails) may be used and the present description should notbe limited to the cell culture media employed in the present Examples.

In some embodiments, different cell expansion methods or cellpopulations described herein may be used together for greater efficacy(e.g., to produce a cell population to be infused or transplanted in asubject to promote accelerated platelet recovery, for example).

In some embodiments, the cells may be propagated for at least 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In someembodiments, the methods of the present description enable propagationof hematopoietic stem cells for at least 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 days, without appreciably losingcell viability and/or “stemness”. As used herein, the expression“appreciably losing cell viability” refers to where the rate of celldeath is greater than the rate of cell expansion, such that a netexpansion of total viable cells of interest is no longer observed. Theperson of skill in the art will recognize that the cell culture mediummay be adapted to suit particular needs of expansion and/ordifferentiation. In some embodiments, the cell culture medium may be anon-differentiating medium.

In some embodiments, mild hyperthermia and addition of a pyrimidoindolederivative agonist of hematopoietic stem cell expansion may be combinedat different points in time and/or for variable lengths of time (e.g.,simultaneously and/or in alternate fashion during a given in vitroculture period), depending on the desired outcome of the culture. Suchvariations are within the scope of some embodiments of the presentdescription, wherein the variations result in an improved amount of cellexpansion relative to the culture of HSCs using only mild hyperthermiaor a pyrimidoindole derivative agonist of hematopoietic stem cellexpansion for a given in vitro culture period.

Accordingly, in some embodiments, the present description relates to amethod for expanding hematopoietic stem cells, and wherein said cellculture medium is a hematopoietic stem cell culture medium. In someembodiments, the methods for expanding hematopoietic stem cells in vitromay be useful for expanding HSCs from cord blood in order to reach asufficient number of cells for successful engraftment in adults. In someembodiments, the present description relates to an in vitro method forexpanding hematopoietic stem cells, the method comprising: (a)propagating said hematopoietic stem cells in a hematopoietic stem cellculture medium comprising a pyrimidoindole derivative agonist ofhematopoietic stem cell expansion; and (b) incubating said hematopoieticstem cells at an incubation temperature between 38° C. and 40° C.,wherein the level of hematopoietic stem cell expansion obtained ishigher than that obtainable by culturing said hematopoietic stem cellsin either (a) or (b) alone. In some embodiments, HSCs are expanded atleast 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, or 1000-fold (e.g., between 5 and 21 days in culture). In someembodiments, the HSCs are long-term-repopulating HSCs (LT-HSCs), such asCD34+/CD45RA− cells, CD34+/CD38−/CD45RA− or CD49f+ cells. In someembodiments, the LT-HSCs are expanded at least 2, 5, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100, or 200-fold (e.g., between 5 and 21 days inculture). The skilled person will understand that the actual values offold expansion obtained may vary depending on a number of factors, suchas the quality and/or quantity of the starting material cells. Thesevariations are within the scope of the present description.

In some embodiments, the present description relates to a method forculturing hematopoietic stem cells to produce myeloid progenitors, andwherein said cell culture medium is a medium promoting differentiationof hematopoietic stem cells towards myeloid progenitors. As used herein,“myeloid progenitor” refers to a hematopoietic cell that is capable ofbeing induced to differentiate into one or more megakaryocytes, one ormore erythrocytes, one or more mast cells or one or more myeloblasts. Insome embodiments, myeloid progenitors may be identified as CD34+/CD41+cells (megakaryocyte progenitors). In some embodiments, the presentdescription relates to an in vitro method for culturing hematopoieticstem cells to produce myeloid progenitors, the method comprising: (a)propagating said hematopoietic stem cells in a cell culture mediumpromoting differentiation of hematopoietic stem cells towards myeloidprogenitors, said cell culture medium comprising a pyrimidoindolederivative agonist of hematopoietic stem cell expansion; and (b)incubating said hematopoietic stem cells at an incubation temperaturebetween 38° C. and 40° C., wherein the level of myeloid progenitorsobtained is higher than that obtainable by culturing said hematopoieticstem cells in either (a) or (b) alone. In some embodiments, the myeloidprogenitors may be promoted to differentiate into megakaryocytes. Insome embodiments, myeloid progenitors are expanded at least 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or1000-fold (e.g., between 5 and 21 days in culture). The skilled personwill understand that the actual values of fold expansion obtained mayvary depending on a number of factors, such as the quality and/orquantity of the starting material cells. These variations are within thescope of the present description.

In some embodiments, the present description relates to a method forculturing hematopoietic stem cells to produce megakaryocytic progenitorcells, and wherein said cell culture medium is a medium promotingdifferentiation of hematopoietic stem cells towards the megakaryocyticlineage. As used herein, “megakaryocytic progenitor” refers to ahematopoietic cell that is capable of being induced to differentiateinto one or more megakaryocytes. In some embodiments, the presentdescription relates to an in vitro method for culturing hematopoieticstem cells to produce megakaryocytic progenitor cells, the methodcomprising: (a) propagating said hematopoietic stem cells in a cellculture medium promoting differentiation of hematopoietic stem cellstowards the megakaryocytic lineage, said cell culture medium comprisinga pyrimidoindole derivative agonist of hematopoietic stem cellexpansion; and (b) incubating said hematopoietic stem cells at anincubation temperature between 38° C. and 40° C., wherein the level ofmegakaryocytic progenitor cells obtained is higher than that obtainableby culturing said hematopoietic stem cells in either (a) or (b) alone.In some embodiments, the megakaryocytic progenitors may be promoted todifferentiate into platelets or platelet-like fragments. In someembodiments, megakaryocytic progenitors are expanded at least 20, 30,40, 50, 60, 70, 80, 90, or 100-fold (e.g., between 5 and 14 days inculture). The skilled person will understand that the actual values offold expansion obtained may vary depending on a number of factors, suchas the quality and/or quantity of the starting material cells. Thesevariations are within the scope of the present description.

In some aspects, the present description relates to the propagation ofcells in the presence of a “pyrimidoindole derivative agonist ofhematopoietic stem cell expansion”. As used herein, this expressionrefers to small-molecule compounds sharing a degree of structuralsimilarity with pyrimidoindole that have the ability to stimulate HSCexpansion. It is understood that by “pyrimidoindole derivative agonistof hematopoietic stem cell expansion”, a sufficient concentration ordose of the compound is present in the cell culture medium to elicit thedesired effect (e.g., stimulate HSC expansion and/or synergize with theuse of mild hyperthermia in the context of the present description). Insome embodiments, the pyrimidoindole derivative may be a“pyrimido[4,5-b]indole derivative”. Such derivatives have beenpreviously described for example in WO 1993/020078; WO 1995/019970; WO1997/002266; WO 1998/042708; WO 2000/066585; WO 2003/037898; WO2004/058764; WO 2005/037825; WO 2006/116733; WO 2008/055233; WO2009/004329, and WO 2010/006032. In some embodiments, thepyrimido[4,5-b]indole derivative may be a compound described for examplein Fares et al., 2014, or WO 2013/110198, such as(1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine[UM171], or methyl4-(3-(piperidin-1-yl)propylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate[UM729]. Such compounds were shown to stimulate expansion ofCD34+/CD45RA− mobilized peripheral blood cells, which are enriched inlong-term-repopulating HSCs (LT-HSCs), without acting as suppressors ofthe aryl hydrocarbon receptor (AhR) pathway (Fares et al., 2014).Accordingly, in some embodiments, the pyrimidoindole derivative mayinclude an AhR pathway-independent pyrimido[4,5-b]indole derivativeagonist of CD34+/CD45RA− cell expansion. In some embodiments, theconcentration the pyrimidoindole derivative agonist of hematopoieticstem cell expansion may be prepared as a stock solution in a carriersuch as DMSO. In some embodiments, the concentration of thepyrimidoindole derivative agonist of hematopoietic stem cell expansionmay be from 10, 15, 20, 25, or 30 nM, to 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 nM. Theconcentration of the pyrimidoindole derivative agonist of hematopoieticstem cell expansion may be modified based on the potency of themolecule, and other concentrations, not explicitly recited here, arealso within the scope of the present description. In some embodiments,pharmaceutically acceptable salts, solvates, prodrugs, or stereoisomersof any of the above-mentioned pyrimidoindole derivatives may also bewithin the scope of the present description. As used herein, the term“prodrug” refers to a compound which, when metabolized (e.g., in vivo),yields the desired active compound. Typically, the prodrug is inactive,or less active than the desired active compound, but may provideadvantageous handling, administration, or metabolic properties. Unlessotherwise specified, a reference to a particular compound also includesprodrugs thereof.

In some embodiments, the pyrimidoindole derivatives of the presentdescription may be used in combination with other compounds, such as anantagonist of aryl hydrocarbon receptor (AhR) (e.g., SR1:4-[2-[[2-benzo[b]thien-3-yl-9-(1-methylethyl)-9H-purin-6-yl]amino]ethyl]-phenol).In some embodiments, the antagonist of aryl hydrocarbon receptorcompound (e.g., SR1) may be prepared as a stock solution in a carriersuch as DMSO. In some embodiments, the antagonist of aryl hydrocarbonreceptor compound (e.g., SR1) may be used for example at between 100 and1000 nM, between 200 and 900 nM, between 300 and 800 nM, between 400 and800 nM, between 450 and 750 nM, between 700 and 800 nM, or between 450and 550 nM.

In some aspects, the present description relates to the propagation ofcells under mild hyperthermia. In some embodiments, “mild hyperthermia”refers to propagating cells at an incubation temperature between 38° C.and 40° C., preferably 39° C. Of course, the skilled person wouldunderstand that some transient variations outside the aforementionedtemperature range may be permitted, as long as the cells are generallycultured/propagated at temperatures 1-3° C. higher than the recommendedtemperature for the given cell population (e.g., generally 37° C.). Insome embodiments, the cells are maintained under mild hyperthermia forat least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,or 21 days. In some embodiments, the cells are maintained under mildhyperthermia for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or 21 consecutive days.

In some aspects, the present description relates to an in vitro expandedcell population which is a population of expanded hematopoietic stemcells, myeloid progenitor cells, megakaryocytic progenitor cells, or anycombination thereof produced by a method defined herein. In someembodiments, different in vitro expanded cell populations of the presentdescription may be combined for greater therapeutic benefit (e.g., topromote accelerated platelet recovery).

In some embodiments, the in vitro expanded cell population of thepresent description may comprise at least 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, or 80% of CD34+/CD41+ cells.

In some aspects, the present description relates to the use of an invitro expanded cell population described herein for transplanting into asubject, or for the manufacture of a therapeutic composition fortransplanting into a subject. In some embodiments, the in vitro expandedcell population may be a population of expanded hematopoietic stem cellsfor use in hematopoietic stem cell transplantation, or for themanufacture of a therapeutic composition for same. In some embodiments,the in vitro expanded cell population may be a population ofmegakaryocytic progenitor cells for use in the treatment ofthrombocytopenia, or for the manufacture of a therapeutic compositionfor same. Accordingly, in some aspects, the present description relatesto a pharmaceutical composition comprising: the population of expandedhematopoietic stem cells, myeloid progenitor cells, or megakaryocyticprogenitor cells, as defined herein.

In some embodiments, the pharmaceutical composition may comprise apharmaceutically acceptable carrier, such as normal buffered saline(e.g., about 135-150 mM NaCl). Other suitable carriers include, but arenot limited to, water, buffered water, 0.4% saline, 0.3% glycine, andthe like. Additional carriers suitable for use in delivering thecultured stem cells of the present invention are described in, e.g.,REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co., Philadelphia,Pa., 18th ed. (1995).

Other objects, advantages and features of the present description willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

EXAMPLES Example 1 Materials and Methods for Examples 2-5 1.1 Materials

All chemicals were purchased from Sigma-Aldrich (Oakville, ON, Canada),unless otherwise noted.

1.2 Isolation and Culture of CD34+ Cells from Human Umbilical Cord Blood

CD34+ cells were isolated from human umbilical cord blood by positiveselection according to standard procedures. Purified CD34+ cells fromeither single units or pooled from several units of cord blood wereseeded in 1 mL of the appropriate culture medium in 24-well plates at adensity of approximately 10⁵ cells/mL. Cells were maintained at adensity of 10⁵-10⁶ cells/mL throughout the culture by dilution and/ormedium replacement.

For culture and differentiation towards the megakaryocytic lineage,purified CD34+ cells were cultured in StemSpan™ ACF medium (STEMCELL™Technologies, Vancouver, BC, Canada) supplemented with antibiotics andthe “BS1” cytokine cocktail described previously (Cortin et al, 2005),and cell counts were performed on days 6, 10, and 14.

For CD34+ cell culture and expansion while limiting differentiation,cells were grown in StemSpan™-ACF medium (STEMCELL™ Technologies)supplemented with StemSpan™ CC110 cytokine cocktail (STEMCELL™Technologies), and counted on days 4, 7, 10, 14, 17, and 21.

Cell counts and viability were measured using the NucleoCounter® NC250™Viability and Cell Count Assay system (ChemoMetec Inc., Davis, Calif.),according to the manufacturer's instructions.

1.3 Cell Phenotype Analyses

Cell phenotypes were determined by flow cytometry on the indicated daysusing the following panel of labeled primary antibodies: CD34-FITC,CD45RA-APC, CD41a-APC, CD42b-PE, and CD235-PE.

1.4 CFU-MK Assay

Aliquots of cells grown in StemSpan™ ACF+BS1 culture medium werecollected on the indicated days, and CFU-MK were determined using theMegacult™ C kit from STEMCELL™ Technologies, according to themanufacturer's instructions.

Example 2 Combination of Culture Under Mild Hyperthermia andPyrimidoindole Compound PIC Increases Expansion of MegakaryocyticProgenitor Cells

CD34+ cells were isolated and cultured for differentiation towards themegakaryocytic lineage as generally described in Example 1.2. Todetermine the effects of the combination of mild hyperthermia and apyrimidoindole compound on megakaryocytic progenitor (MKP) cellexpansion, the CD34+ cells were cultured at either 37° C. or 39° C., andin the presence of cell culture medium supplemented or not with 35 nM(final concentration) of the pyrimidoindole compound:(1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diaminehydrochloride (“UM171”) herein referred to as “PIC”. Flow cytometry wasused to monitor the expansion of CD34+/CD41+ MKP cells over time, asdescribed in Example 1.3.

As shown in FIG. 1, cells cultured at 37° C. in the presence (“37°C.+PIC”) or absence (“37° C.”) of PIC had less than 5-fold expansion ofCD34+/CD41+ MKP cells, as did cells cultured under mild hyperthermia inthe absence of PIC (“39° C.”). Interestingly and surprisingly, thecombination of mild hyperthermia and PIC (“39° C.+PIC”) resulted in anexpansion of CD34+/CD41+ MKP cells that reached nearly 100-fold by day14, with a purity of CD34+/CD41+ cells generally above 40% (data notshown). These results show a potent synergistic effect of mildhyperthermia combined with a pyrimidoindole compound in stimulatingexpansion of MKP cells from CD34+ cells.

Example 3 Combination of Culture Under Mild Hyperthermia andPyrimidoindole Compound PIC Yields a More Homogeneous Population ofMegakaryocytic Progenitor Cells

More thorough cell phenotypic profiles of the CD34+ cells isolated andcultured in Example 2 were determined on days 6, 10 and 14 by flowcytometry as described in Example 1.3. The results are summarized inTable 1, which shows the percentage of cells bearing the indicated cellsurface marker or combination of markers.

TABLE 1 Phenotypic analysis of CD34+ cells cultured in conditionsfavoring MKP cell expansion Percentage of cells bearing cell surfacemarker(s) Day of CD34+/ CD34+/ CD41+/ culture Conditions CD34+ CD41−CD41+ CD42+ CD45RA+ CD235+ CD41+ CD42+ Day 6 37° C. 80.82 47.29 42.5015.41 71.94 19.75 31.21 15.13 37° C. + PIC 98.38 68.55 29.27 2.48 71.5511.67 29.54 1.71 39° C. 27.93 8.89 72.30 34.93 62.37 35.89 34.93 34.8439° C. + PIC 97.19 48.02 49.84 8.31 58.97 19.51 48.85 7.95 Day 10 37° C.29.11 15.92 61.10 39.22 35.09 15.86 13.26 39.05 37° C. + PIC 84.39 64.4024.84 2.55 76.43 7.45 20.21 1.30 39° C. 16.84 5.44 89.13 74.36 15.5715.64 15.17 74.07 39° C. + PIC 87.29 21.37 73.31 17.30 46.94 14.40 67.1017.09 Day 14 37° C. 21.41 4.44 81.52 44.49 34.55 18.53 14.49 44.41 37°C. + PIC 69.53 54.97 23.65 3.02 61.90 2.90 11.01 0.68 39° C. 13.8 2.4888.23 69.6 27.68 36.8 10.1 68.05 39° C. + PIC 88.56 10.25 87.90 27.0927.09 12.61 76.46 27.04

As shown in Table 1, culturing CD34+ cells under mild hyperthermia andin the presence of PIC (“39° C.+PIC”) resulted in a greater proportionof CD34+/CD41+ MKP cells on days 6, 10 and 14, than culturing CD34+cells at a standard temperature in the absence (“37° C.”) or presence(“37° C.+PIC”) of PIC, or than culturing the cells under mildhyperthermia without PIC (“39° C.”). More particularly, CD34+ cellscultured for 14 days under mild hyperthermia and in the presence of PIC(“39° C.+PIC”) yielded 76.5% of CD34+/CD41+ MKP cells, whereas the useof PIC alone (“37° C.+PIC”) or mild hyperthermia alone (“39° C.”)yielded only 11% and 10% of CD34+/CD41+ MKP cells, respectively. Theseresults show that a more homogeneous cellular population ofmegakaryocytic progenitors can be obtained by culturing CD34+hematopoietic stem cells in standard conditions favoring differentiationinto megakaryocytic progenitors, wherein the standard culture conditionsare modified by the combination of a pyrimidoindole compound and anincubation temperature of 39° C.

Furthermore, as shown in Table 1, cultures grown at 39° C. in thepresence of PIC (“39° C.+PIC”) only have 27% of cells of phenotypeCD41+/CD42+ typical of mature megakaryocytes on day 14, whereas culturesgrown at 39° C. in the absence of PIC (“39° C.”) have 68% of CD41+/CD42+mature megakaryocytes. Cultures grown at 37° C. in the presence of PIC(“37° C.+PIC”) have virtually no CD41+/CD42+ mature megakaryocytes onday 14, whereas cultures grown at 37° C. in the absence of PIC (“37°C.”) have 44% of CD41+/CD42+ mature megakaryocytes. These resultssuggest that the pyrimidoindole compound blocks the hyperthermia-induceddifferentiation of CD34+ cells before the mature CD41+/CD42+megakaryocyte stage, and/or preferentially expands and maintainsCD34+/CD41+ megakaryocytic progenitors. Accordingly, in someembodiments, the pyrimidoindole compound may be removed and the cellsmay be further propagated under conditions of mild hyperthermia, therebyproducing a more synchronized cell population.

Without being bound by theory, the distribution of the cell phenotypesobtained when mild hyperthermia and PIC were combined strongly suggeststhat both effectors act in synergy to push towards and maintain theCD34+ cell differentiation towards the megakaryocytic lineage up to theprogenitor level, so that after 14 days of culture, CD34+/CD41+ MKPsrepresent the majority of the cells in the entire population.

Example 4 Combination of Culture Under Mild Hyperthermia andPyrimidoindole Compound Enhances the Number of CFU-MKs

Starting with purified CD34+ cells isolated and cultured as described inExample 2, the effect of a 39° C. incubation temperature and of the PICpyrimidoindole compound, alone or in combination, on the number ofCFU-MK obtained, was analyzed by a standard progenitor cell assay asdescribed in Example 1.4.

As shown in FIG. 2, the combined effect of a 39° C. incubationtemperature and PIC (“39° C.+PIC”) produced a greater number of CFU-MK,as compared to either condition tested individually (“39° C.” or “37°C.+PIC”). More particularly, the number of CFU-MK obtained for “39°C.+PIC” was more than twice that obtained when the culture was grown at37° C. with PIC (“37° C.+PIC”), and more than four times the number ofCFU-MK obtained when the culture was grown at 39° C. in the absence ofPIC (“39° C.”). Furthermore, the number of CFU-MK obtained from cellscultured at 39° C. with PIC (“39° C.+PIC”) was greater than thatobtainable by adding the number of CFU-MKs of either condition testedindividually (“39° C.” and “37° C.+PIC”). These results show that thecombination of mild hyperthermia and PIC synergized so as to increasethe number of CFU-MK obtained. This unforeseen effect is particularlyinteresting, since current technologies are inefficient at expandingcells with CFU-MK potential in vitro.

Example 5 Combination of Culture Under Mild Hyperthermia andPyrimidoindole Compound PIC Increases Expansion of CD34+ Cells whileMaintaining their Primitive Phenotype

CD34+ cells were isolated and cultured in a medium suitable formaintaining their self-renewal while limiting their differentiation intoprogenitors (StemSpan™ ACF supplemented with StemSpan™ CC110 cytokinecocktail, as described in Example 1.2). The CD34+ cells were cultured ateither 37° C. or 39° C., in the presence of a cell culture mediumsupplemented or not with 35 nM (final concentration) of thepyrimidoindole compound PIC.

As shown in FIG. 3(A), cells cultured at 37° C. without PIC (“37° C.”)yielded modest expansion of CD34+ cells at any time during the culture.Supplementing the cell culture medium with PIC increased the expansionto 100-200—fold on day 21. Strikingly, while cells cultured at 39° C.without PIC (“39° C.”) yielded minimal cellular expansion of CD34+cells, culturing the cells at 39° C. in the presence of PIC (“39°C.+PIC”) resulted in a dramatic increase in CD34+ cell expansion, withmore than a 1000-fold expansion on day 21. These results show a potentsynergistic effect of mild hyperthermia and a pyrimidoindole compound onthe expansion of CD34+ cells.

Cell phenotypic profiles of the expanded CD34+ cells were determined ondays 4, 7, 10, 14, 17, and 21 by flow cytometry as described in Example1.3. The results are summarized in Table 2, which shows the percentageof cells bearing the indicated cell surface marker or combination ofmarkers.

TABLE 2 Phenotypic analysis of CD34+ cells cultured in conditionsfavoring CD34+ cell expansion with limited differentiation Percentage ofcells bearing cell surface marker(s) Day of CD34+/ CD34+/ CD34+/ cultureConditions CD34+ CD41+ CD42+ CD45RA+ CD45RA− CD235+ CD41+ CD235+ Day 437° C. 94.30 8.14 2.32 68.21 5.44 7.67 5.10 37° C. + PIC 97.91 12.531.08 65.34 4.87 12.51 4.83 39° C. 85.81 7.92 4.16 62.68 8.62 5.03 5.9439° C. + PIC 94.74 9.05 2.21 57.92 4.11 8.37 3.65 Day 7 37° C. 80.3813.62 5.13 68.46 9.42 12.10 8.18 37° C. + PIC 94.61 15.93 1.30 80.075.42 15.74 5.28 39° C. 85.29 10.83 13.94  95.42 13.94  9.44 13.00  39°C. + PIC 88.27 18.12 2.92 74.14 10.89  16.17 9.68 Day 10 37° C. 34.4612.95 8.58 79.08 5.41 8.80 2.82 37° C. + PIC 84.26 14.19 0.48 88.37 2.3312.94 2.16 39° C. —* —* —* —* —* —* —* —* 39° C. + PIC 71.28 27.40 1.3582.34 9.36 20.80 7.94 Day 14 37° C. 20.49 8.14 3.61 71.95  5.26 4.094.37 1.90 37° C. + PIC 58.28 15.16 0.19 84.63 15.22 2.10 9.55 1.39 39°C. —* —* —* —* —* —* —* —* 39° C. + PIC 48.58 32.04 1.16 81.97 19.745.13 13.88 3.72 Day 17 37° C. 13.03 9.43 1.84 76.57  4.38 5.24 2.30 2.0437° C. + PIC 48.29 24.70 0.63 87.89 13.58 3.94 9.75 0.52 39° C. —* —* —*—* —* —* —* —* 39° C. + PIC Day 21 37° C. 37° C. + PIC 39° C. —* —* —*—* —* —* —* —* 39° C. + PIC *N/D: Not determined.

The results of the phenotypic analysis presented in Table 2 indicatethat the combined treatment of mild hyperthermia and PIC did not lead toan enhanced differentiation of the cells towards the megakaryocytic anderythroid (CD235+) lineages, which suggests that “stemness” wasmaintained to some extent. Without being bound by theory, these resultscollectively suggest that mild hyperthermia combined with the use of apyrimidoindole compound preserves the anti-differentiation effects ofthe pyrimidoindole compound, while simultaneously potentiating thestimulatory effects of the 39° C. incubation temperature on the in vitroexpansion of the cells.

The results presented in FIG. 3(A) are derived from the entirepopulation of CD34+ cells. A minor population of CD34+/CD45RA− cells arethought to represent genuine long-term hematopoietic stem cells(LT-HSCs). Combining the results of total cell expansion and those ofTable 2 allowed to determine the absolute numbers and fold expansion ofCD34+/CD45RA− LT-HSCs. As shown in FIG. 3(B), the expansion ofCD34+/CD45RA− LT-HSCs was potently enhanced by culturing cells at 39° C.in the presence of the PIC compound (“39° C.+PIC”). This resultdemonstrates that the synergistic effect of mild hyperthermia and apyrimidoindole agonist of hematopoietic stem cell expansion could beexploited for a variety of purposes and for the derivation of severalhematopoietic lineages.

Example 6 Materials and Methods for Examples 7-16 6.1 Sources of CD34+Cells

Human cord blood (CB) was collected after obtaining written informedconsent from donors, following our institutional Research EthicsCommittee guidelines. Mononuclear cells (MNCs) were first separated overa Ficoll-Hypaque™ density gradient (GE Healthcare), then cryopreservedat −180° C. in Cryostor CS10™ medium (STEMCELL Technologies). ThawedMNCs from six to eight CB were pooled before CD34 isolation. CB CD34+cells were enriched by positive selection using the EasySep™ CD34enrichment kit, according to the manufacturer's instructions (STEMCELLTechnologies).

Human bone marrow and G-CSF-mobilized peripheral blood CD34+ cells werepurchased from AllCells.

6.2 Culture and Differentiation of CD34+-Enriched Cells Towards theMegakaryocytic Lineage

Human CD34+-enriched cells (purity ≥90%) were seeded in 24-well platesat 100 000 cells/mL in expansion medium consisting of either (1)StemSpan™ ACF (ACF; STEMCELL Technologies) supplemented with the OMPCcytokine cocktail (Robert et al., 2011), or (2) StemSpan™ SFEM (STEMCELLTechnologies) supplemented with the BS1 megakaryocyte expansion anddifferentiation cocktail (Cortin et al., 2005). OMPC consists of 35ng/mL thrombopoietin (TPO; Feldan Therapeutics), 10 ng/mL stem cellfactor (SCF; Peprotech), and 11 ng/mL human FMS-like tyrosine kinase 3ligand (FLT3, Peprotech). BS1 consists of 1 ng/mL SCF, 30 ng/mL TPO, 7.5ng/mL IL-6 and 13.5 ng/mL IL-9 (Feldan Therapeutics).

Cultures were maintained in a humidified atmosphere with 5% CO₂ at 37°C. or 39° C., as indicated in the Examples. Cells were diluted at 300000 cells/mL with fresh medium every 3 to 4 days. Stock solutions of PICand PIC2 were prepared by dissolving in DMSO, then added directly tocultured cells at the following final effective concentrations: PIC: 35nM (see Example 2); PIC2: 500 nM (see Example 7.4).

6.3 Culture and Differentiation of CD34+-Enriched Cells Towards theErythroid Lineage

Human CB CD34+-enriched cells (purity ≥90%) were seeded in 24-wellplates at 100 000 cells/mL in expansion medium consisting of Eave'sbasal medium (Iscove's modified Dulbecco's medium (IMDM), 20% BIT (10ng/mL bovine serum albumin, 10 μg/mL bovine pancreatic insulin, 200μg/mL human transferrin; STEMCELL Technologies), 0.1 mg/mL low-densitylipoprotein (STEMCELL Technologies), 50 μM 2-mercaptoethanol)supplemented with 20 ng/mL SCF (Peprotech) and 2 U/mL erythropoietin(EPO; Feldan Therapeutics). Cultures were maintained in a humidifiedatmosphere with 5% CO₂ at 37° C. or 39° C., as indicated in theExamples. Cells were diluted at 300 000 cells/mL with fresh medium every3 to 4 days. A stock solution of PIC was prepared by dissolving in DMSO,then added directly to cultured cells at a final effective concentrationof 35 nM.

6.4 Culture of CD34+-Enriched Hematopoietic Stem Cells in ConditionsFavoring Self-Renewal

Human CD34+-enriched cells (purity ≥90%) were seeded in 24- or 96-wellplates at 500 000 cells/mL in one of three expansion media consisting ofStemSpan™ ACF (ACF), SFEM, or SFEM II (STEMCELL Technologies),supplemented with either CC110 (STEMCELL Technologies) or a home-made(HM) cytokine cocktail consisting of 100 ng/mL human FLT3 (Peprotech),100 ng/mL SCF (Peprotech), 50 ng/mL TPO (Feldan Therapeutics) and 10μg/mL low-density lipoprotein (LDL, STEMCELL Technologies). Cultureswere maintained in a humidified atmosphere with 5% CO₂ at 37° C. or 39°C., as indicated in the Examples. Cells were diluted at 500 000 cells/mLwith fresh medium every 3 to 4 days. Stock solutions of PIC and PIC2were prepared by dissolving in DMSO, then added directly to culturedcells at the following final effective concentrations: PIC: 35 nM; PIC2:500 nM.

6.5 Analysis of Cultured Cells

Cellular counts and viability were determined by adding Solution 18(Acridine Orange and DAPI solution, ChemomMetec) and usingNucleoCounter™ NC-250 for detection (ChemomMetec).

For cells expanded in conditions favoring differentiation towards the MKlineage, surface markers were detected using the following antibodies:CD34-FITC, CD41a-APC and CD42b-PE. All antibodies were purchased from BDBiosciences, except for CD34-FITC which was purchased from Immunotech.7AAD was used as a viability dye. All samples were analyzed on anAccuri™ C6 flow cytometer (BD Biosciences), and raw data were analyzedwith FCS Express 5 Flow Research Edition software from at least 15 000viable cell events acquired for each sample. Colony-forming-unitmegakaryocytes (CFU-MK) were assayed using MegaCult-C™, according to themanufacturer's instructions (STEMCELL Technologies).

For cells expanded in conditions favoring differentiation towards theerythroid lineage, surface markers were detected using the followingantibodies: CD34-FITC, CD71-APC and CD235-PE. All antibodies werepurchased from BD Biosciences, except for CD34-FITC which was purchasedfrom Immunotech. 7AAD was used as a viability dye. All samples wereanalyzed on an Accuri™ C6 flow cytometer (BD Biosciences), and raw datawere analyzed with FCS Express 5 Flow Research Edition software from atleast 15 000 viable cell events acquired for each sample.

For cells expanded in conditions favoring self-renewal, surface markerswere detected using the following antibodies: CD34-PE, CD45RA-FITC, andCD38-BV421. All antibodies were purchased from BD Biosciences, exceptfor CD34-PE which was purchased from Immunotech. Labeling of aldehydedehydrogenase (ALDH)-positive cells was performed using the ALDEFLUOR™Kit (STEMCELL Technologies), according to the manufacturer'sinstructions. 7AAD was used as a viability dye. All samples wereanalyzed on a FACS-Cy Flow ML system (Sysmex), and raw data wereanalyzed with FCS Express 5 Flow Research Edition software from at least15 000 viable cell events acquired for each sample.

6.6 Transplantation of CD41+ Cells into Mice, and Evaluation of BoneMarrow Engraftment

Seven to nine weeks old female NOD.Cg-Prkdc^(scid) II2rg^(tm1Wjl)/SzJ(NSG) mice were purchased from The Jackson Laboratory (Bar Harbor, Me.).Sublethally irradiated mice (250 cGy, ¹³⁷Cs) were transplantedintravenously by tail vein injection with CB CD34+-enriched cellsexpanded in StemSpan™ SFEM medium supplemented with the BS1 cocktail for10 days. Each experiment included mice injected with PBS as negativecontrol for bone marrow engraftment. Experimental and control groups forevaluation of bone marrow (BM) engraftment and human platelet productionconsisted of at least 6 mice. Engraftment of human cells in the bonemarrow of mice was evaluated between 12 to 16 weeks post-transplantationby flushing femurs and tibias and analyzing freshly isolated BM cells byflow cytometry using the following antibodies: human CD45-PE-Cy7, mouseCD45-PE, human CD41-APC, human CD33-APC, human CD34-PE, human CD235-PE,human CD3-FITC. 7AAD was used as a viability dye. Red cells were lysedusing BD Lysing solution following the manufacturer's instructions.

6.7 Analysis of Human Platelet Production in Transplanted Mice

Retro-orbital venous plexus blood was collected from anesthetized miceusing EDTA-coated capillaries (Drummond). The evaluation of humanplatelet production in transplanted mice consisted of two distinctsteps. First, murine platelet counts were determined by staining murineplatelets with a rat anti-mouse CD41-FITC antibody in whole blood.Samples were then brought to a final dilution of 1/18 000 in PBS/1% BSA,and the concentration of murine platelets was assayed by flow cytometryusing the BD Accuri™ C6 instrument. Second, the proportion of humanplatelets was measured in platelet-rich plasma (PRP). PRP was preparedby diluting whole blood in PBS (½) and centrifuging for 30 seconds at800 RPM. Ten microliters of PRP was stained with mouse anti-humanCD41-APC and rat anti-mouse CD41-FITC antibodies. Samples were thenanalyzed on the BD Accuri™ C6 instrument; at least 400 000 events wereacquired in the platelet region of the dot-plot.

6.8 Transplantation into Mice of Fresh CB CD34+ Cells, or ProgeniesThereof Obtained by In Vitro Culture of CB CD34+ Cells in ConditionsFavoring Self-Renewal, and Evaluation of Bone Marrow Engraftment

Seven to nine weeks old female NOD.Cg-Prkdc^(scid) II2rgm^(tm1Wjl)/SzJ(NSG) mice were purchased from The Jackson Laboratory (Bar Harbor, Me.).Sublethally irradiated mice (250 cGy, ¹³⁷Cs) were transplantedintravenously by tail vein injection with either fresh CD34+-enriched CBcells, or their total cell progeny that had been in vitro-expanded for12 days. Each experiment included mice injected with PBS as negativecontrol for bone marrow engraftment. Experimental and control groupsconsisted of at least 5 mice. Engraftment of human cells in the bonemarrow of mice was evaluated at 27 weeks post-transplantation byflushing femurs and tibias and analyzing freshly isolated BM cells byflow cytometry using the following antibodies: human CD45-PE-Cy7, mouseCD45-PE, human CD33-BV421, human CD19-FITC, human CD34-PE, and humanCD3-FITC. 7AAD was used as a viability dye.

Example 7 Effect of Different Sources of CD34+ Cells on Expansion ofCD34+/CD41+ Megakaryocytic Progenitors Under Mild Hyperthermia andPyrimidoindole Compounds

7.1 Combined Use of Mild Hyperthermia and PIC Results in SynergisticExpansion of Megakaryocytic Progenitors from Cord Blood CD34+ Cells

Human CD34+-enriched cells were obtained from human cord blood (CB) asdescribed in Example 6.1, and were cultured in StemSpan™ ACF mediumsupplemented with the OMPC cytokine cocktail as described in Example 6.2in the presence or absence of mild hyperthermia (39° C.) and/or thepresence or absence of the pyrimidoindole compound PIC (see Example 2).Cellular counts and viability were determined as described in Example6.5.

FIG. 4 shows the fold expansion of CD34+/CD41+ cells (megakaryocyticprogenitors) under the different culture conditions tested over 14 days.These results show a synergistic effect of the combined use of mildhyperthermia (39° C.) and PIC on the expansion of CD34+/CD41+ cells fromcord blood CD34+ cells.

7.2 Combined Use of Mild Hyperthermia and PIC Results in SynergisticExpansion of Megakaryocytic Progenitors from Mobilized Peripheral BloodCD34+ Cells

Human G-CSF-mobilized peripheral blood CD34+ cells were obtained asdescribed in Example 6.1, and were cultured in StemSpan™ SFEM mediumsupplemented with the BS1 megakaryocyte expansion and differentiationcocktail as described in Example 6.2 in the presence or absence of mildhyperthermia (39° C.) and/or the presence or absence of thepyrimidoindole compound PIC (see Example 2). Cellular counts andviability were determined as described in Example 6.5.

FIG. 5 shows the fold expansion of CD34+/CD41+ cells (megakaryocyticprogenitors) under the different culture conditions tested over 14 days.These results show a synergistic effect of the combined use of mildhyperthermia (39° C.) and PIC on the expansion of CD34+/CD41+ cells frommobilized peripheral blood CD34+ cells.

7.3 Combined Use of Mild Hyperthermia and PIC Results in SynergisticExpansion of Megakaryocytic Progenitors from Bone Marrow CD34+ Cells

Human bone marrow CD34+ cells were obtained as described in Example 6.1,and were cultured in StemSpan™ SFEM medium supplemented with the BS1megakaryocyte expansion and differentiation cocktail as described inExample 6.2 in the presence or absence of mild hyperthermia (39° C.)and/or the presence or absence of the pyrimidoindole compound PIC (seeExample 2). Cellular counts and viability were determined as describedin Example 6.5.

FIG. 6 shows the fold expansion of CD34+/CD41+ cells (megakaryocyticprogenitors) under the different culture conditions tested over 14 days.These results show a synergistic effect of the combined use of mildhyperthermia (39° C.) and PIC on the expansion of CD34+/CD41+ cells frombone marrow CD34+ cells.

7.4 Combined Use of Mild Hyperthermia and PIC2 Results in SynergisticExpansion of Megakaryocytic Progenitors from Cord Blood CD34+ Cells

Human CD34+-enriched cells were obtained from human cord blood (CB) asdescribed in Example 6.1, and were cultured in StemSpan™ SFEM mediumsupplemented with the BS1 cytokine cocktail as described in Example 6.2in the presence or absence of mild hyperthermia (39° C.) and/or thepresence or absence of the pyrimidoindole compound “PIC2”:

-   -   Methyl 4-((3-(piperidin-1-yl)propyl)amino)-9H-pyrimido[4,5-b]        indole-7-carboxylate (“UM729”).

Cellular counts and viability were determined as described in Example6.5.

FIG. 7 shows the fold expansion of CD34+/CD41+ cells (megakaryocyticprogenitors) under the different culture conditions tested over 14 days.These results show a synergistic effect of the combined use of mildhyperthermia (39° C.) and PIC2 on the expansion of CB CD34+/CD41+ cells.

Example 8 Production of Human Platelets in Mice Following Infusion of InVitro-Expanded MK Progenitors

Human CD41+ cells were prepared by expanding CB CD34+ cells in vitrounder mild hyperthermia and in conditions favoring the preferentialexpansion of MK progenitors, as described in Example 6.2; StemSpan™ SFEMmedium was supplemented with the BS1 cytokine cocktail for the in vitroexpansion. Measured doses of CD41+ cells obtained after 10 days ofculture were transplanted into NSG mice as described in Example 6.6.

Human platelet production in mice was determined by counting humanplatelets in murine blood either five days or 2.5 weeks post-infusion ofCB CD34+ cells expanded in vitro in conditions favoring the preferentialexpansion of MK progenitors, as described in Example 6.2. FIGS. 8 and 9show the results of short-term human platelet production in mice at 5days and 2.5 weeks, respectively, wherein “MK-6M”: infusion of sixmillion CD41+ cells produced using StemSpan™ SFEM+BS1 cocktail;“(MK+PIC)-1M”: infusion of one million CD41+ cells produced usingStemSpan™ SFEM+BS1 cocktail supplemented with PIC; “(MK+PIC)-6M”:infusion of six million CD41+ cells produced using StemSpan™ SFEM+BS1cocktail supplemented with PIC; and “PBS”: infusion ofphosphate-buffered saline as a control. The numbers to the right of thedash symbols represent mean numbers of human platelets/μL of blood foreach condition.

Example 9 Effect of Erythroid Differentiation Medium on Expansion ofCD34+ Cells Under Mild Hyperthermia and PIC

Human CD34+-enriched cells were obtained from human cord blood (CB) asdescribed in Example 6.1, and were cultured in an erythroiddifferentiation medium as described in Example 6.3 in the presence orabsence of mild hyperthermia (39° C.) and/or the presence or absence ofthe pyrimidoindole compound PIC (see Example 2). Cellular counts andviability were determined as described in Example 6.5.

9.1 Effect of PIC on Expansion of CD71+ Cells (Erythroid Precursors) inErythroid Differentiation Medium

FIG. 10 shows the fold expansion of CB CD71+ cells (erythroidprecursors) under the different culture conditions tested over 14 days.The use of mild hyperthermia (39° C.) resulted in an increase in theexpansion of CD71+ erythroid precursors (see FIG. 10, “37” vs. “39”, and“37+“PIC” vs. “39+PIC”). However, the addition of PIC resulted in adecrease in the expansion of CD71+ erythroid precursors (see FIG. 10,“37” vs. “37+PIC”, and “39” vs. “39+PIC”).

FIG. 11 shows the fold expansion of total cells (total cell expansion)under the different culture conditions tested over 14 days. The use ofmild hyperthermia (39° C.) resulted in an increase in total cellexpansion (see FIG. 11, “37” vs. “39”, and “37+PIC” vs. “39+PIC”).However, the addition of PIC resulted in a decrease in total cellexpansion (see FIG. 11, “37” vs. “37+PIC”, and “39” vs. “39+PIC”).

9.2 Additive Effect of Mild Hyperthermia and PIC on Expansion of CD34+Cells (HSCs) and CD34+/CD71+ Cells (Erythroid Progenitors) in ErythroidDifferentiation Medium

FIGS. 12 and 13 show the fold expansion of CD34+ cells and CD34+/CD71+cells (erythroid progenitors), respectively, under the different cultureconditions tested over 14 days. Interestingly, these results show thatthe use of mild hyperthermia (39° C.) and PIC have an additive effect onthe expansion of both CB CD34+ cells (FIG. 12) and CB CD34+/CD71+ cells(erythroid progenitors) (FIG. 13) in an erythroid differentiationmedium.

Example 10 Effect of Different Cytokine Cocktails on Expansion of CD34+Cells (HSCs) and CD34+/CD45RA− Cells (LT-HSCs) Under Mild Hyperthermiaand PIC

The results presented in Example 5 showed that the combined use of mildhyperthermia (39° C.) and PIC resulted in an increase in expansion ofCD34+ cells while limiting their differentiation when using StemSpan™ACF medium supplemented with the commercially available CC110 cytokinecocktail. The results presented in this example show that synergisticexpansion of CB CD34+ HSCs under mild hyperthermia (39° C.) and PIC canbe obtained using a home-made (HM) cytokine cocktail.

Human CB CD34+-enriched cells were obtained from human cord blood (CB)as described in Example 6.1, and were cultured in conditions favoringtheir self-renewal as generally described in Example 6.4, in thepresence or absence of mild hyperthermia (39° C.) and/or the presence orabsence of the pyrimidoindole compound PIC (see Example 2). Cellularcounts and viability were determined as described in Example 6.5.

FIG. 14 shows the fold-expansion of CB CD34+ cells (hematopoietic stemcells) cultured in StemSpan™ ACF medium supplemented with either theCC110 cytokine cocktail (FIG. 14A) or the home-made (HM) cytokinecocktail (FIG. 14B).

FIG. 15 shows the fold-expansion of CB CD34+/CD45RA− (long-termhematopoietic stem cells; LT-HSCs) cells cultured in StemSpan™ ACFmedium supplemented with either the CC110 cytokine cocktail (FIG. 15A)or the home-made (HM) cytokine cocktail (FIG. 15B).

Table 3 shows the percentages of each of the indicated HSCsubpopulations after culture of CB CD34+ cells in StemSpan™ ACF mediumsupplemented with either the home-made (HM) or CC110 cytokine cocktailfor 14 days.

TABLE 3 Phenotypic analysis of CD34+ cells cultured in StemSpan ™ ACFmedium supplemented with either the HM or CC110 cytokine cocktailPercentages of each of the indicated phenotypes Cytokine CD34+/ CD34+/CD34+/CD38−/ cocktail Condition CD34+ CD45RA− CD38− CD45RA− CD38−CD45RA−/ HM 37° C. 15.24 42.8 15.6 4.5 7.9 1.97 37° C. + PIC 51.5 53.412.05 22.4 32.4 14.7 39° C. 23.1 43.1 15.1 7.9 10.4 3.13 39° C. + PIC40.95 35.2 12 24.8 22.9 15.7 CC110 37° C. 18.5 49 18.8 6.5 9.25 2.69 37°C. + PIC 48.9 59.2 14.8 16.36 30.3 10 39° C. 23 50.15 16.3 7.9 10.4 3.4339° C. + PIC 43.7 40.85 14.45 23.05 24.2 14.1

Example 11 Effect of Different Basal Media and Cytokine Cocktails onExpansion of CD34+ Cells (HSCs) and CD34+/CD45RA− Cells (LT-HSCs) UnderMild Hyperthermia and PIC

The results presented in this example show that synergistic expansion ofCB CD34+ HSCs and CD34+/CD45RA− LT-HSCs under mild hyperthermia (39° C.)and PIC can be obtained using different commercially available media anddifferent cytokine cocktails.

Human CB CD34+-enriched cells were obtained from human cord blood (CB)as described in Example 6.1, and were cultured in conditions favoringtheir self-renewal as generally described in Example 6.4, in thepresence or absence of mild hyperthermia (39° C.) and/or the presence orabsence of the pyrimidoindole compound PIC (see Example 2). Cellularcounts and viability were determined as described in Example 6.5.

FIG. 16 shows the fold-expansion of CB CD34+ cells (hematopoietic stemcells) cultured in StemSpan™ SFEM medium supplemented with either theCC110 cytokine cocktail (FIG. 16A) or the home-made (HM) cytokinecocktail (FIG. 16B).

FIG. 17 shows the fold-expansion of CB CD34+ cells (hematopoietic stemcells) cultured in StemSpan™ SFEM II medium supplemented with either theCC110 cytokine cocktail (FIG. 17A) or the home-made (HM) cytokinecocktail (FIG. 17B).

FIG. 18 shows the fold-expansion of CB CD34+ cells (hematopoietic stemcells) cultured in StemSpan™ ACF medium supplemented with either theCC110 cytokine cocktail (FIG. 18A) or the home-made (HM) cytokinecocktail (FIG. 18B). FIG. 18C shows the fold-expansion of CBCD34+/CD45RA− cells (long-term hematopoietic stem cells; LT-HSC)cultured in StemSpan™ SFEM medium supplemented with the BS1 cytokinecocktail under mild hyperthermia and in the presence (“39”) or absence(“CTL”) of PIC.

Table 4 shows the fold expansion of long-term hematopoietic stem cells(LT-HSCs) (CD34+/CD45RA−) from CB CD34+ cells cultured in various basalmedia using the CC110 or the home-made (HM) cytokine cocktail.

TABLE 4 Expansion of CD34+/CD45RA− cells (LT-HSCs) cultured in variousbasal media and cytokine cocktails Fold expansion on the Medium +indicated days of culture cytokine cocktail Condition Day 7 Day 10 Day14 Day 18 SFEM + CC110 37° C. 5.65 8.6 11.3 17.9 37° C. + PIC 4.14 6.714.2 27.1 39° C. 5.06 9.2 18.6 26.9 39° C. + PIC 7.01 19.8 67.8 129.4SFEM + HM 37° C. 4.35 8.8 8.8 13.8 37° C. + PIC 4.46 13.0 16.2 24.8 39°C. 6.44 11.5 16.9 24.8 39° C. + PIC 6.12 19.3 53.7 122.3 SFEM II + CC11037° C. 6.38 6.8 7.7 22.1 37° C. + PIC 5.52 10.3 15.0 24.3 39° C. 6.939.2 14.6 31.9 39° C. + PIC 9.45 35.4 139.8 237.6 SFEM II + HM 37° C.4.57 6.0 27.7 18.8 37° C. + PIC 4.18 9.7 18.3 31.7 39° C. 7.28 9.5 63.329.8 39° C. + PIC 6.97 25.9 99.6 208.3 ACF + CC110 37° C. 3.99 5.9 8.615.4 37° C. + PIC 6.56 13.4 25.9 47.6 39° C. 4.96 10.0 18.0 33.1 39°C. + PIC 9.16 34.8 104.8 191.5 ACF + HM 37° C. 4.38 6.2 11.9 17.1 37°C. + PIC 6.51 28.8 20.6 43.8 39° C. 4.43 8.7 10.8 21.5 39° C. + PIC10.64 34.0 131.6 330.4

Table 5 shows the percentages of each of the indicated HSCsubpopulations derived from CB CD34+ cells cultured for 14 days invarious basal media (SFEM, SFEM II, or StemSpan ACF (ACF)) mediumsupplemented with the CC110 or home-made (HM) cytokine cocktail.

TABLE 5 Phenotypic analysis of CD34+ cells cultured in different basalmedia and in different cytokine cocktails Percentages of each of theindicated phenotypes Medium + CD34+/ cytokine cocktail Condition CD34+CD45RA− CD45RA− SFEM + CC110 37° C. 18.6 66.99 4.41 37° C. + PIC 42.6878.72 6.96 39° C. 18.94 58.78 4.72 39° C. + PIC 45.36 70.13 12.23 SFEM +HM 37° C. 14.67 63.1 3.68 37° C. + PIC 37.4 75.74 6.66 39° C. 16.5957.67 4.16 39° C. + PIC 41.95 69.88 12.39 SFEM II + CC110 37° C. 9.6559.53 1.9 37° C. + PIC 33.38 77.73 6.09 39° C. 12.64 55.23 2.41 39° C. +PIC 37.9 63.9 15.03 SFEM II + HM 37° C. 11.55 60.78 9.08 37° C. + PIC30.82 77.27 6.11 39° C. 12.4 56.26 10.48 39° C. + PIC 37.56 63.45 15.27ACF + CC110 37° C. 15.37 57.36 3.7 37° C. + PIC 50.98 76.09 11.83 39° C.19.94 59.6 5.14 39° C. + PIC 43.73 59.72 18.73 ACF + HM 37° C. 17.3959.16 4.84 37° C. + PIC 44.62 77.44 7.67 39° C. 17.13 59.92 3.9 39° C. +PIC 44.82 59.67 17.68

FIG. 18C shows the fold-expansion of CB CD34+/CD45RA− cells (long-termhematopoietic stem cells; LT-HSC) cultured in StemSpan™ SFEM mediumsupplemented with the BS1 cytokine cocktail. These results show that acell culture medium known for promoting expansion of megakaryocyticprogenitor cells (CD34+/CD41+), also enables expansion of self-renewingLT-HSCs.

Example 12 Effect of the Combined Use of Mild Hyperthermia and PIC2 orPIC on Expansion of CD34+ Cells (HSCs), CD34+/CD45RA− Cells (LT-HSCs),and CD34+/CD38-/CD45RA− Cells

Human CB CD34+-enriched cells were obtained from human cord blood (CB)as described in Example 6.1, and were cultured in conditions favoringtheir self-renewal as generally described in Example 6.4 in StemSpan™ACF medium supplemented with the home-made (HM) cytokine cocktail, inthe presence or absence of mild hyperthermia (39° C.) and/or thepresence or absence of the pyrimidoindole compound PIC2 (see Example7.4). Cellular counts and viability were determined as described inExample 6.5.

FIGS. 19, 20 and 21 show the fold-expansion of CD34+ cells(hematopoietic stem cells), CD34+/CD45RA− cells (LT-HSCs), andCD34+/CD38−/CD45RA− cells (Majeti et al., 2007), respectively, after 7,10, and 14 days in culture.

FIG. 22 shows the fold-expansion of CD34+/CD38−/CD45RA− cells after 14days of culture under mild hyperthermia (39° C.) and PIC on theexpansion of CB CD34+/CD38−/CD45RA− cells cultured for 14 days inStemSpan™ ACF medium supplemented with the home-made (HM) cytokinecocktail. Data represent means of four independent experiments.

Example 13 Effect of the Combined Use of Mild Hyperthermia and PIC onthe Expansion of Mobilized Peripheral Blood (mPB) CD34+(HSCs),CD34+/CD45RA− (LT-HSCs), and CD34+/CD38−/CD45RA− Cells

Human G-CSF-mobilized peripheral blood CD34+-enriched cells wereobtained as described in Example 6.1, and were cultured in conditionsfavoring their self-renewal as generally described in Example 6.4 inStemSpan™ ACF medium supplemented with the home-made (HM) cytokinecocktail, in the presence or absence of mild hyperthermia (39° C.)and/or the presence or absence of the pyrimidoindole compound PIC (seeExample 2). Cellular counts and viability were determined as describedin Example 6.5.

FIGS. 23, 24 and 25 show the fold-expansion of CD34+ cells(hematopoietic stem cells), CD34+/CD45RA− cells (LT-HSCs), andCD34+/CD38−/CD45RA− cells, respectively, after 10, 14, and 17 days inculture.

Example 14 Effect of the Combined Use of Mild Hyperthermia and PIC onthe Expansion of Bone Marrow CD34+(HSCs), CD34+/CD45RA− (LT-HSCs), andCD34+/CD38-CD45RA− Cells

Human bone marrow (BM) CD34+-enriched cells were obtained as describedin Example 6.1, and were cultured in conditions favoring theirself-renewal as generally described in Example 6.4 in StemSpan™ ACFmedium supplemented with the home-made (HM) cytokine cocktail, in thepresence or absence of mild hyperthermia (39° C.) and/or the presence orabsence of the pyrimidoindole compound PIC (see Example 2). Cellularcounts and viability were determined as described in Example 6.5.

FIGS. 26, 27 and 28 show the fold-expansion of CD34+ cells(hematopoietic stem cells), CD34+/CD45RA− cells (LT-HSCs), andCD34+/CD38−/CD45RA− cells, respectively, after 10, 14, and 17 days inculture.

Example 15 Effect of the Combined Use of Mild Hyperthermia and PIC onthe Expansion of CB CD34+/ALDH^(Bright) Cells

Human CB CD34+-enriched cells were obtained from human cord blood (CB)as described in Example 6.1, and were cultured in conditions favoringtheir self-renewal as generally described in Example 6.4 in StemSpan™ACF medium supplemented with the home-made (HM) cytokine cocktail, inthe presence or absence of mild hyperthermia (39° C.) and/or thepresence or absence of the pyrimidoindole compound PIC (see Example 2).Cellular counts and viability were determined as described in Example6.5.

FIG. 29 shows the fold-expansion of CD34+/ALDH^(Bright) cells after 21days of culture. These results indicate that the combined use of mildhyperthermia and PIC results in a synergistic expansion of thissubpopulation of HSCs.

Example 16 Transplantation and Engraftment in Immunodeficient Mice ofHuman CB CD34+ Cells (HSCs) Expanded In Vitro Under Mild Hyperthermiaand PIC

Human CB CD34+-enriched cells were obtained from human cord blood (CB)as described in Example 6.1, and were cultured in conditions favoringtheir self-renewal as generally described in Example 6.4 in StemSpan™ACF medium supplemented with the home-made (HM) cytokine cocktail in thepresence of mild hyperthermia (39° C.) and the pyrimidoindole compoundPIC (see Example 2). Fresh CB CD34+ cells, or CB CD34+ cells that hadbeen expanded for 12 days, were transplanted into immunodeficient miceas described in Example 6.8.

An increase in the percentage of human CD45+ cells in the bone marrow ofmice transplanted with the in vitro expanded CB CD34+ cells wasobserved, as compared to the mice transplanted with PBS alone,indicating successful engraftment (data not shown).

REFERENCES

-   Cortin et al., (2005). Efficient in vitro megakaryocyte maturation    using cytokine cocktails optimized by statistical experimental    design. Exp Hematol 33 (10): 1182-1191.-   Fares et al., (2014). Cord blood expansion. Pyrimidoindole    derivatives are agonists of human hematopoietic stem cell    self-renewal. Science 345 (6203): 1509-1512.-   Majeti et al., (2007). Identification of a hierarchy of multipotent    hematopoietic progenitors in human cord blood. Cell Stem Cell 1 (6):    635-645.-   Proulx et al., (2004). Increased megakaryopoiesis in cultures of    CD34-enriched cord blood cells maintained at 39 degrees C.    Biotechnol Bioeng 88 (06): 675-680.-   Robert et al., (2011). Glycoprotein Ibα receptor instability is    associated with loss of quality in platelets produced in culture.    Stem Cells and Development 20 (03): 379-390.

1-21. (canceled)
 22. An in vitro method for expanding hematopoietic stemcells (HSCs), said method comprising: (a) propagating said HSCs in acell culture medium comprising a pyrimidoindole derivative agonist ofhematopoietic stem cell expansion; and (b) incubating said HSCs at anincubation temperature between 38° C. and 40° C., wherein said cellculture medium is a hematopoietic stem cell culture medium that enablespropagation of HSCs under self-renewal conditions, and wherein the levelof HSC expansion obtained is higher than that obtainable by culturingsaid HSCs in either (a) or (b) alone.
 23. The method of claim 22,wherein: (i) said HSCs are CD34+ hematopoietic stem cells; and/or (ii)said HSCs are from: umbilical cord blood; bone marrow; peripheral blood;induced pluripotent stem cells; embryonic stem cells;transdifferentiated from differentiated cells of non-hematopoieticorigin; genetically modified hematopoietic stem cells; immortalizedhematopoietic stem cells; other sources of pluripotent or multipotentcells; or any combination thereof.
 24. The method of claim 23, whereinsaid HSCs are from mobilized peripheral blood cells, and/or are fromresidual cells following leukoreduction, deleukocytation, and/or otherblood purification or processing of peripheral blood.
 25. The method ofclaim 23, wherein said HSCs are from unmobilized peripheral blood cells.26. The method of claim 22, wherein said HSCs are incubated in a cellculture medium comprising the pyrimidoindole derivative agonist ofhematopoietic stem cell expansion, and at an incubation temperaturebetween 38° C. and 40° C., for at least 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 days.
 27. The method of claim 22,wherein said incubation temperature is 39° C.
 28. The method of claim22, wherein said pyrimidoindole derivative agonist of hematopoietic stemcell expansion is: (1) a pyrimido[4,5-b]indole derivative; (2)(1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine;(3) methyl4-(3-(piperidin-1-yl)propylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate;(4) methyl4-(3-(piperidin-1-yl)propylamino)-9H-pyrimido[4,5-b]indole-7-carboxylatehydrochloride; (5) a pharmaceutically acceptable salt, solvate, prodrug,or stereoisomer of any one of (a) to (d); or (6) any combination of (1)to (5).
 29. The method of claim 22, wherein said cell culture mediumcomprises: (i) stem cell factor (SCF); thrombopoietin (TPO); or both SCFand TPO; or (ii) human FMS-like tyrosine kinase 3 ligand (FLT3); stemcell factor (SCF); thrombopoietin (TPO); low-density lipoprotein (LDL);or any combination thereof.
 30. The method of claim 22, furthercomprising: (c) removing said pyrimidoindole derivative agonist ofhematopoietic stem cell expansion and continuing to propagate said HSCsat an incubation temperature between 38° C. and 40° C. or at anincubation temperature of about 37° C.
 31. The method of claim 22,wherein the method results in expansion of CD34+/CD45RA− long-termhematopoietic stem cells.
 32. An in vitro method for producingmegakaryocytic progenitor cells, said method comprising: (a) propagatinghematopoietic stem cells (HSCs) in a cell culture medium comprising apyrimidoindole derivative agonist of hematopoietic stem cell expansion;and (b) incubating said HSCs at an incubation temperature between 38° C.and 40° C., wherein said cell culture medium is a medium promotingdifferentiation of hematopoietic stem cells towards the megakaryocyticlineage, and wherein the level of megakaryocytic progenitor cellsproduced is higher than that obtainable by culturing said HSCs in either(a) or (b) alone.
 33. The method of claim 32, wherein: (i) said HSCs areCD34+ hematopoietic stem cells; and/or (ii) said HSCs are from:umbilical cord blood; bone marrow; peripheral blood; induced pluripotentstem cells; embryonic stem cells; transdifferentiated fromdifferentiated cells of non-hematopoietic origin; genetically modifiedhematopoietic stem cells; immortalized hematopoietic stem cells; othersources of pluripotent or multipotent cells; or any combination thereof.34. The method of claim 33, wherein said HSCs are from mobilizedperipheral blood cells, and/or are from residual cells followingleukoreduction, deleukocytation, and/or other blood purification orprocessing of peripheral blood.
 35. The method of claim 33, wherein saidHSCs are from unmobilized peripheral blood cells.
 36. The method ofclaim 32, wherein said HSCs are incubated in a cell culture mediumcomprising the pyrimidoindole derivative agonist of hematopoietic stemcell expansion, and at an incubation temperature between 38° C. and 40°C., for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or 21 days.
 37. The method of claim 32, wherein said incubationtemperature is 39° C.
 38. The method of claim 32, wherein saidpyrimidoindole derivative agonist of hematopoietic stem cell expansionis: (1) a pyrimido[4,5-b]indole derivative; (2)(1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine;(3) methyl4-(3-(piperidin-1-yl)propylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate;(4) methyl4-(3-(piperidin-1-yl)propylamino)-9H-pyrimido[4,5-b]indole-7-carboxylatehydrochloride; (5) a pharmaceutically acceptable salt, solvate, prodrug,or stereoisomer of any one of (a) to (d); or (6) any combination of (1)to (5).
 39. The method of claim 32, wherein said cell culture mediumcomprises: stem cell factor (SCF); thrombopoietin (TPO); human FMS-liketyrosine kinase 3 ligand (FLT3); IL-6; IL-9; or any combination thereof.40. The method of claim 32, further comprising: (c) removing saidpyrimidoindole derivative agonist of hematopoietic stem cell expansionand continuing to propagate said HSCs at an incubation temperaturebetween 38° C. and 40° C. or at an incubation temperature of about 37°C.
 41. The method of claim 32, wherein the method results in theproduction of a population of megakaryocytic progenitor cells comprisingat least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of CD34+/CD41+cells.